Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of the kidney epithelial cell line MDCK with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a large increase in the permeability of tight junctions within 2-4 hr. This change in transepithelial permeability is accompanied by alterations in cell morphology that include the opening of tight junctions, loss of apical surface microvilli and the formation of cytoplasmic processes containing numerous microtubules, microfilaments and 10 nm intermediate filaments. Simultaneously, TPA also stimulates a 10-20 fold increase in the synthesis and secretion of the serine protease plasminogen activator (PA); however, the inhibition of PA synthesis by cycloheximide does not prevent the morphological changes or the increased tight junctional permeability, suggesting that PA is not involved in these early TPA-induced events. Drugs that inhibit cytoskeletal function, such as cytochalasins B and D, colchicine, and vinblastine sulfate, are all relatively effective in preventing the TPA-induced morphological changes but not the increases in transepithelial permeability. Our results provide evidence that possible sites of TPA action include epithelial tight junctions as well as components of the cytoskeleton.
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PMID:Tumor promoter-induced changes in the permeability of epithelial cell tight junctions. 719 50

Endothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (+/- 3000), 43,000 (+/- 2000), 26,000 (+/- 1500) and 14,500 (+/- 1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivatives by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60 degrees C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.
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PMID:Secretion of plasminogen activators by cultured bovine endothelial cells: partial purification, characterization and evidence for multiple forms. 719 62

Experiments were initiated to gain an understanding of the environmental factors that may regulate injury-induced mitosis and wound healing in the mammalian lens. The addition of thrombin or trypsin to a completely defined serum-free medium stimulated cell proliferation and migration in the cultured mammalian lens. A 30 min exposure of the rabbit lens to highly purified thrombin induced DNA synthesis and mitosis throughout the normally amitotic central region of the lens epithelium. Lenses exposed to thrombin for 24 or 52 hr exhibited cell migration and mitosis. The mitotic response brought about by thrombin was totally curtailed by hirudin and antithrombin III. Prothrombin, papain, or pepsin were not mitogenic toward the cultured lens. A 30 min exposure of the lens to trypsin induced cell division and migration, a response that did not occur in the presence of trypsin inhibitors. Lenses cultured in a trypsin-containing medium for 24 hr showed extensive cell death throughout the entire central region of the epithelium. In addition, an endogenous serine protease, plasminogen activator, was detected in cultured rabbit lens epithelial cells. Wound healing in the lens in vivo is accompanied by cellular migration and mitosis. The present experiments demonstrate that a highly purified serine protease, thrombin, which is present at the site of lenticular injury in vivo, is capable of inducing mitosis and migration in lens epithelia. The results suggest that thrombin or other exogenous and endogenous serine proteases might contribute to the process of wound healing in the ocular lens.
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PMID:Thrombin induces cell division in rabbit lenses cultured in a completely defined serum-free medium. 719 16

Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains. From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned. The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation. To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA. The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography. All molecules were expressed in their single-chain form and could be converted to their two-chain form. With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied. All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose. Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator. 747 91

BM 06.022 is a tissue-type plasminogen activator deletion variant that is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive inclusion bodies in E. coli and transferred into the active enzyme by an in vitro folding process. Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active centers of both enzymes are rather similar. The further evaluation of the site of interaction of BM 06.022 and DnsEGRck by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound selectively to His322, which is part of the catalytic triad of this serine protease.
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PMID:Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone demonstrates the full activity of the refolded and purified tissue-type plasminogen activator variant BM 06.022. 749 32

We have analyzed the proteolytic activity of a recombinant form of apolipoprotein(a) [r-apo-(a)]. A mutant 17-kringle from of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (tPA) as determined by SDS-PAGE and fluorography and by Western blot analysis. However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated into r-Lp(a) particles, were uniformly inactive against a variety of chromogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteolytic activity, we generated truncated forms of the Ser-->Arg proteolytic activity, we generated truncated forms of the Ser-->Arg mutant containing one or 10 kringle IV repeats. These truncated versions of r-apo(a) were susceptible to cleavage by tPA but were inactive against the plasmin substrate S-2251. Treatment of the Ser-->Arg mutant of the 17-kringle r-apo(a) with tPA and diisopropylflurophosphate (DFP) did not result in modification of the mutant protease domain by DFP. Finally, we incubated r-apo(a) or r-Lp(a) particles formed in vitro with purified human LDL; no degradation of LDL was observed after 16 h at 37 degrees C. The results of this study suggest that one or more of the substitutions present in the protease domain of apo(a), in addition to the Arg-->Ser substitution, render apo(a) proteolytically inactive.
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PMID:Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a). 749 9

Unlike most proteases, tissue-type plasminogen activator (t-PA) is not synthesized as an inactive precursor or zymogen. Instead, the single-chain "proenzyme" form of t-PA possesses very significant catalytic activity. Recent investigations of the molecular basis of the unusually high enzymatic activity of single-chain t-PA have focused attention upon Asp-194, a residue that is invariant among chymotrypsin-like enzymes. The critical role of this residue in securing the active conformation of mature chymotrypsin-like enzymes has been discussed extensively. Subsequent work, however, has indicated that this conserved residue can also form interactions that dramatically influence the catalytic activity of serine protease zymogens. While Asp-194 forms interactions that suppress the activity of the zymogen chymotrypsinogen, it may, by contrast, directly promote the catalytically active conformation of single-chain t-PA. To test the hypothesis that Asp-194 promotes the activity of both single- and two-chain t-PA and therefore plays opposing roles in single-chain t-PA and chymotrypsinogen, and also to examine whether this invariant residue plays an essential role in the stimulation of t-PA by fibrin, we used site-directed mutagenesis to construct the following variants of t-PA: t-PA/D194E, t-PA/D194N, t-PA/R15E,D194E, and t-PA/R15E,D194N. In the absence of fibrin, the activity of enzymes carrying a mutation at position 194 was reduced by factors of 1000-2000 compared to wild type t-PA. Similar reductions of activity were observed for both single- and two-chain variants, suggesting an important role for Asp-194 in both forms of the enzyme. The mutated enzymes, however, displayed a dramatically enhanced response to fibrin monomers. While the activity of wild type t-PA was stimulated by fibrin monomers by a factor of 960, the corresponding stimulation factor for the mutated enzymes varied from 498,000-1,050,000.
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PMID:Variants of tissue-type plasminogen activator with substantially enhanced response and selectivity toward fibrin co-factors. 755 5

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.
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PMID:Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways. 755 92

Tissue-type plasminogen activator (t-PA) is a serine protease, catalyzing the initial step in the fibrinolytic process. Intravenously administered t-PA is rapidly cleared from the circulation by the liver. Two distinct clearance mechanisms, which are mediated by the low density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and by the mannose receptor on liver endothelial cells, have been described. Using competitors and inhibitors of the receptors, we investigated the role of LRP and carbohydrate receptors in t-PA clearance in vivo. To inhibit LRP, the 39-kD protein, which is a potent inhibitor of LRP activity, was overexpressed in the liver of mice using an adenoviral gene transfer technique. Expression of the 39-kD protein resulted in a sustained plasma concentration and an increase in the plasma half-life of 125I-t-PA from less than 1 min to 4-5 min. Blockade of the mannose receptor by intravenous administration of ovalbumin also prolonged the plasma half-life of 125I-t-PA to 3-4 min. The same degree of inhibition of t-PA clearance was also observed after administration of an inhibitor of the fucose receptor, fucosyl-BSA. However, under the conditions established for the complete blockade of the mannose receptor, no additional inhibition of t-PA clearance was observed using fucosyl-BSA, suggesting little or no role for the fucose receptor in the clearance of t-PA. Furthermore, a dramatic increase of the plasma half-life of 125I-t-PA (>> 20 min) was observed in mice overexpressing 39-kD protein and administered ovalbumin +/- fucosyl-BSA. Our results clearly demonstrate that two independent receptor systems, LRP and the mannose receptor, are involved in the hepatic clearance of t-PA.
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PMID:Two receptor systems are involved in the plasma clearance of tissue-type plasminogen activator (t-PA) in vivo. 763 54

The plasminogen activator (PA)/plasmin pathway has been implicated in a variety of physiologic and pathologic processes that require tissue remodeling and cell motility. The pathway is highly regulated and results in the generation of the broad spectrum serine protease, plasmin, from the zymogen plasminogen. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are produced by osteoblasts, as are the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) and a cellular receptor for uPA. Little is known about regulation of the receptor, but the other 3 components of the pathway are regulated differentially by various osteotropic hormones and local factors. Several roles have been proposed for this pathway in bone. These involve proteolytic activation of procollagenase and latent growth factors, such as transforming growth factor beta (TGF beta) and insulin-like growth factor (IGF), as well as cell motility as a result of pericellular proteolysis. The roles of this pathway in bone remodeling may not be confined to proteolytic events, because uPA has been reported to act as a mitogen on osteoblast-like cells. This effect is independent of proteolytic activity but requires the growth factor domain of uPA to bind to uPA cellular receptors. If the plasminogen activator/plasmin pathway encompasses these roles, it would serve to couple formation and resorption of bone in a highly regulated manner.
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PMID:The plasminogen activator inhibitor system in bone cell function. 764 98


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